A Retinoid X Receptor (RXR)-Selective Retinoid Reveals That RXR-Α Is

Home , Retinoid receptor, Retinoid X receptor

Available online http://breast-cancer-research.com/content/6/5/R546

ResearchVol 6 No 5 article Open Access A retinoid X receptor (RXR)-selective retinoid reveals that RXR-α is potentially a therapeutic target in breast cancer cell lines, and that it potentiates antiproliferative and apoptotic responses to peroxisome proliferator-activated receptor ligands David L Crowe1 and Roshantha AS Chandraratna2

1Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California, USA 2Division of Retinoid Research, Allergan, Irvine, California, USA

Corresponding author: David L Crowe, [email protected]

Received: 2 Nov 2003 Revisions requested: 13 Jan 2004 Revisions received: 15 Jun 2004 Accepted: 24 Jun 2004 Published: 23 Jul 2004

Breast Cancer Res 2004, 6:R546-R555 (DOI 10.1186/bcr913)http://breast-cancer-research.com/content/6/5/R546 © 2004 Crowe et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

Abstract

Introduction Certain lipids have been shown to be ligands for a Methods We determined the effects of selective PPAR and subgroup of the nuclear hormone receptor superfamily known RXR ligands on established human breast cancer cell lines in as the peroxisome proliferator-activated receptors (PPARs). vitro. Ligands for these transcription factors have been used in experimental cancer therapies. PPARs heterodimerize and bind Results PPAR-α and PPAR-γ ligands induced apoptotic and DNA with retinoid X receptors (RXRs), which have homology to antiproliferative responses in human breast cancer cell lines, other members of the nuclear receptor superfamily. Retinoids respectively, which were associated with specific changes in have been found to be effective in treating many types of cancer. gene expression. These responses were potentiated by the However, many breast cancers become resistant to the RXR-selective ligand AGN194204. Interestingly, RXR-α- chemotherapeutic effects of these drugs. Recently, RXR- overexpressing retinoic acid resistant breast cancer cell lines selective ligands were discovered that inhibited proliferation of were more sensitive to the effects of the RXR-selective all-trans retinoic acid resistant breast cancer cells in vitro and compound. caused regression of the disease in animal models. There are few published studies on the efficacy of combined therapy using Conclusion RXR-selective retinoids can potentiate the PPAR and RXR ligands for breast cancer prevention or antiproliferative and apoptotic responses of breast cancer cell treatment. lines to PPAR ligands.

Keywords: cell cycle, chemotherapy, hormones, nuclear receptors, transcription factors

Introduction PPARs have functional domains for DNA and ligand bind- Within the past few years, specific lipids have been shown ing [4]. PPARs bind to recognition sequences in the pro- to be ligands for a subgroup of the nuclear hormone recep- moter regions of their target genes to regulate tor superfamily known as the peroxisome proliferator-acti- transcription. These sequences typically contain a direct vated receptors (PPARs; for review, see Mandrup and Lane repeat of the motif PuGGTCA separated by one nucle- [1]). The first of these ligand-dependent transcription fac- otide; this sequence is recognized by other members of the tors, namely PPAR-α, was originally cloned as an orphan nuclear hormone receptor superfamily, such as retinoid X receptor that was activated by agents that induce peroxi- receptors (RXRs) [5]. These receptors exhibit markedly dif- some proliferation in the liver (for review, see Keller and ferent tissue distributions, DNA binding affinities, and lig- Wahli [2]). Subsequently, PPAR-δ (also known as PPAR-β) and preferences. PPARs are activated by long-chain fatty and PPAR-γ were cloned by low stringency screening [3]. acids (for review, see Kliewer and coworkers [6]). PPAR-α Like other members of the nuclear receptor superfamily, can be selectively activated by hydroxyeicosatetraenoic

BrdU = bromodeoxyuridine; Cdk = cyclin-dependent kinase; ERK = extracellular signal-regulated kinase; HETE = hydroxyeicosatetraenoic acid; LA = γ-linolenic acid; PBS = phosphate-buffered saline; PPAR = peroxisome proliferator-activated receptor; RA = retinoic acid; RAR = retinoic acid receptor; RXR = retinoid X receptor; TUNEL = dUTP nick-end labeling; TZD = thiazolidinedione drug. R546 Breast Cancer Research Vol 6 No 5 Crowe and Chandraratna

acid (HETE) and fibrates, whereas PPAR-γ preferentially The effects of PPAR and RXR ligands on established binds γ-linolenic acid (LA), prostaglandin J2 metabolites, breast cancer cell lines require further study. The evaluation and thiazolidinedione drugs (TZDs) [6,7]. of new RXR ligands that cooperate with PPARs may result in important therapeutic breakthroughs. To provide a Given the ability of PPARs to induce differentiation of a vari- molecular and cell biologic basis for these studies, we ety of cell types, ligands for these transcription factors have examined the effects of selective PPAR and RXR ligands been used in experimental cancer therapies (for review, see on established breast cancer cell lines in vitro. We deter- Roberts-Thomson [8]). TZD administration to liposarcoma mined that the RXR-selective ligand potentiated the anti- patients induced histologic and biochemical differentiation proliferative and apoptotic responses to PPAR ligands in markers in vivo [9]. TZD treatment of human bladder carci- human breast cancer cell lines. RA-resistant lines that over- noma cells in vitro inhibited cellular proliferation via expressed RXR-α were more sensitive to the effects of increased cyclin-dependent kinase (Cdk) inhibitor expres- these ligands, suggesting potential therapeutic uses for sion and induced cell death [10]. Similarly, the growth of these compounds. human lung cancer cell lines was inhibited via apoptosis when cultures were treated with TZDs but not the PPAR-γ Methods agonist bezafibrate [11]. TZDs also induced differentiation Cell culture and stable transfection of human colon cancer cell lines in vitro with concomitant The human breast cancer cell lines used in the present growth arrest [12]. Loss of function mutations in PPARs study were purchased from the American Type Culture that impaired ligand binding have also been found in colon Collection. Cells were cultured in Dulbecco's modified cancer [13]. Human breast cancer cell lines express Eagle's medium, 10% fetal bovine serum, and 40 µg/ml PPARs [14], and fatty acids were able to stimulate activa- gentamicin at 37°C in a humidified atmosphere of 5% car- tion of a reporter gene via PPAR-γ [15,16]. As seen in other bon dioxide. T47D cells were transfected with 5 µg expres- cancers, TZDs induced growth arrest and differentiation in sion vector for RXR-α (kindly provided by Dr Ronald Evans) breast carcinoma cells in vitro and in animal models or neomycin resistance plasmid alone using Lipofectamine [17,18]. However, a small phase II study of the PPAR-γ lig- reagent (Invitrogen, Carlsbad, CA, USA), in accordance and troglitazone failed to identify objective responses in with the manufacturer's recommendations. Cells were patients with refractory breast cancer [19]. selected in 400 µg/ml G418 for 14 days. Resistant clones were picked for expansion and characterization. RXR-α PPARs heterodimerize and bind DNA with RXRs, which expression was determined by western blot (see below). have homology to other members of the nuclear receptor superfamily (for review, see Mangelsdorf and Evans [20]). Cell death and proliferation assays The natural ligand for RXRs is 9-cis retinoic acid (RA) [21]. The dUTP nick-end labeling (TUNEL) assay for in situ cell RXR heterodimers typically bind to the 5' half of the DNA death detection was performed using a commercially avail- direct repeat and may also homodimerize [22]. However, in able kit (Roche Molecular Biochemicals, Indianapolis, IN, PPAR/RXR heterodimers, the retinoid receptor occupies USA). Cells were treated with 0.1% dimethyl sulfoxide vehi- the 3' half of the response element [5]. Heterodimerization cle, 100 µmol/l HETE, 100 µmol/l LA, or 100 nmol/l of the greatly enhances DNA binding and transcriptional activa- RXR-selective retinoid AGN194204. Cells were fixed in tion [23,24]. RXR overexpression has been reported to 4% paraformaldehyde (pH 7.4) and permeabilized with enhance the transcriptional response to ligand binding 0.1% Triton X-100, 0.1% sodium citrate for 2 min on ice. A [25]. mouse IgM anti-human Fas antibody (Molecular Biology Laboratories, Woburn, MA, USA) that induces apoptosis in Retinoids have been effective in vitro against many types of sensitive cell lines was used as the positive control. An iso- cancer, including that of the breast [26-30]. However, type matched control antibody was used as the negative many breast cancers become resistant to the chemothera- control. After washing with phosphate-buffered saline peutic effects of these drugs [31-33]. Recently, RXR-selec- (PBS), cells were incubated with fluorescein-dUTP and ter- tive ligands were discovered that inhibited proliferation of minal deoxynucleotidyl transferase for 60 min at 37°C, in all-trans RA resistant breast cancer cells in vitro and accordance with the manufacturer's recommendations caused regression of the disease in animal models [34]. (Roche Molecular Biochemicals). After washing three times These RXR-selective ligands did not produce the side in PBS, apoptotic cells were visualized by fluorescence effects that are normally associated with retinoid chemo- microscopy. Proliferation was analyzed by plating 5 × 104 therapy [35]. However, a phase II trial using one of these cells in triplicate cultures and treating with vehicle, PPAR, ligands (bexarotene) showed limited response in patients or RXR selective ligands for up to 6 days. Cells were with metastatic breast cancer [36]. Interestingly, a high-risk trypsinized and counted with a hemacytometer at 2-day lesion for invasive breast cancer, ductal carcinoma in situ, intervals. has been shown to overexpress RXR-α [37]. R547 Available online http://breast-cancer-research.com/content/6/5/R546

To determine G1 to S phase progression, bromodeoxyurid- Transient transfection and reporter gene assays ine (BrdU) incorporation analysis was performed. Vehicle Human breast cancer cell lines were plated in triplicate into and ligand treated cells were incubated with 10 µmol/l six-well tissue culture plates. Cells were transiently trans- BrdU for 1 hour, washed in PBS, and fixed in 70% ethanol fected with 5 µg of a heterologous promoter containing a containing 50 mmol/l glycine (pH 2) for 20 min at -20°C. direct repeat of the PPAR recognition sequence (5'-AGGT- After washing in PBS, the cells were incubated with mouse CAGAGGTCA-3') cloned in the pGL3 luciferase reporter anti-BrdU primary antibody for 30 min at 37°C, in accord- vector (Promega, Madison, WI, USA) using Lipofectamine ance with the manufacturer's recommendations (Roche (Invitrogen), in accordance with the manufacturer's recom- Molecular Biochemicals). The cells were washed in PBS mendations. β-Galactosidase expression vector 1 µg was and incubated with anti-mouse IgG secondary antibody used to normalize for transfection efficiency. Cells were conjugated to fluorescein. Following extensive washing in treated with vehicle, 100 µmol/l LA, 100 nmol/l all-trans PBS, the number of BrdU-positive cells were visualized by RA, 100 nmol/l AGN194204, or a combination of LA plus fluorescence microscopy and expressed as a percentage RA or LA plus AGN194204 for 24 hours following trans- of the total cells counted in 10 random high-power fields. fection. Reporter gene activity was determined using a commercially available kit (Dual-light; Tropix, Foster City, Western blot CA, USA). Luciferase activity was normalized to β-galac- Total protein (75 µg) from vehicle or ligand treated cells tosidase levels for each sample. was separated by SDS-PAGE on 10% resolving gels under denaturing and reducing conditions. Separated pro- Results teins were electroblotted to polyvinylidene difluoride mem- Expression of functional PPAR and RXR proteins branes, in accordance with the manufacturer's To determine the expression of PPARs, RARs, and RXRs in recommendations (Bio-Rad, Hercules, CA, USA). Blots human breast cancer cells, we analyzed six lines by west- were incubated with anti-human primary antibodies to ern blot. As shown in Fig. 1a, all human breast cancer cell PPARs, retinoic acid receptors (RARs), RXRs, cyclins, lines expressed low levels of PPAR-α. Expression of PPAR- Cdks, epidermal growth factor receptor, c-met, extracellu- α was sixfold higher in Hs578T cells than in the other five lar signal-regulated kinases (ERKs), p38, c-myc, AP-1 pro- lines. Relative expression of PPAR-γ was considerably teins, caspases, and β-actin (Transduction Laboratories, higher than that of PPAR-α; however, PPAR-γ expression San Diego, CA, USA and Santa Cruz Biotechnology, Santa was threefold lower in MCF7 and Hs578T cells than in Cruz, CA, USA) for 16 hours at 4°C. After washing in Tris- other lines. RAR-α protein was detected in all cell lines with buffered saline containing 0.1% Tween 20 (pH 7.5), blots the exception of T47D, and expression was generally lower were incubated for 30 min at room temperature with anti- in RA-resistant lines such as MDA-MB-231 and MDA-MB- IgG secondary antibody conjugated to horseradish peroxi- 468. RAR-β expression was below the limit of detection for dase. After extensive washing in Tris-buffered saline con- western blot with the exception of the T47D and SKBR3 taining 0.1% Tween 20 at room temperature, bands were lines, in which low levels of the protein were detected. visualized by the enhanced chemiluminescence method RXR-α expression was low or undetectable in most lines, (Roche Molecular Biochemicals). but it was threefold higher in MDA-MB-468 and SKBR3 cells. We did not detect expression of other PPAR, RAR, or Immunoprecipitation RXR proteins in these cell lines. We concluded that PPARs Cells were lysed in 50 mmol/l HEPES (pH 7.5), 150 mmol/ and RAR-α proteins were expressed in human breast can- l NaCl, 1 mmol/l EDTA, 2.5 mmol/l EGTA, 1 mmol/l DTT, cer cell lines, with higher RXR-α levels in two out of four 1% Nonidet P-40, 10% glycerol, and protease inhibitors for estrogen receptor negative lines. 30 min at 4°C. Lysates were centrifuged at 10,000 g for 10 min and anti-human primary antibody to RXR-α (Santa Cruz PPARs have been shown to heterodimerize with RXRs to Biotechnology) was incubated with the supernatants for 1 activate gene transcription [20]. To determine whether hour at 4°C. Antigen–antibody complexes were precipi- PPAR-α and PPAR-γ could dimerize with RXR-α in human tated by incubation with protein A/G agarose (Santa Cruz breast cancer lines, we immunoprecipitated the retinoid Biotechnology) for 1 hour at 4°C. Immunoprecipitated pro- receptor from these cells. As shown in Fig. 1b, RXR-α proteins were washed three times with 1 ml lysis buffer. Immu- tein could be detected by the more sensitive immunopre- noprecipitated protein complexes were separated by SDS- cipitation technique in all cell lines tested. To determine PAGE as described above. Blots were incubated with anti- whether PPARs associated with RXR-α in these lines, we PPAR antibodies for 16 hours at 4°C. Blots were stripped incubated these blots with anti-PPAR-α and PPAR-γ anti- and incubated with anti-RXR-α antibody to determine bodies. Both PPARs immunoprecipitated with RXR-α pro- amounts of immunoprecipitated protein in each lane. tein in all cell lines. These results indicate that PPARs and RXR-α interact in human breast cancer cell lines.

R548 Breast Cancer Research Vol 6 No 5 Crowe and Chandraratna

Figure 1 To determine whether these heterodimers could activate a PPAR-responsive promoter, we transiently transfected four breast cancer cell lines with a heterologous PPAR promoter vector and determined reporter gene activity in response to selective ligands. As shown in Fig. 1c, treatment with 100 µmol/l LA (a selective PPAR-γ ligand) resulted in threefold to fourfold induction of promoter activity in all cell lines. Treatment with 100 nmol/l of the RXR- selective compound AGN194204 produced twofold to threefold increases in promoter activity in these lines. How- ever, treatment with the RAR agonist all-trans RA failed to induce promoter activity, indicating the specificity of this promoter construct. Treatment of transiently transfected breast cancer cell lines with LA and RA simultaneously did not induce activity above that observed with LA alone. However, combining LA with AGN194204 resulted in an eightfold induction of promoter activity in MCF7 and T47D lines, and 15- to 17-fold induction in MDA-MB-468 and SKBR3 cells, which express higher levels of RXR-α. These results indicate that PPAR/RXR heterodimers are functional in human breast cancer lines, and that RXR-selective ligands can potentiate PPAR-induced transcriptional activation in cells with higher levels of RXR-α.

PPAR-γ/RXR synergy results in antiproliferative effects on human breast cancer cell lines These findings suggest that the RXR-selective ligand AGN194204 may be more biologically active against RXR- α overexpressing breast cancer cell lines. To examine this possibility, we evaluated the proliferative response of this compound as a single agent or in combination with PPAR- selective ligands. As shown in Fig. 2a, treatment with 100 nmol/l AGN194204 slightly inhibited proliferation of T47D cells. The PPAR-γ selective ligand LA was a more effective antiproliferative agent in vitro, producing 30% inhibition of Expressionreceptors (RXRs)(PPARs), of functionally in retinoichuma ninteract breastacid receptorsing cancer peroxisome cell (RARs), lines proliferator-activated and retinoid X cell growth in these assays. Simultaneous treatment with receptors (PPARs), retinoic acid receptors (RARs), and retinoid X receptors (RXRs) in human breast cancer cell lines. (a) Whole cell both ligands resulted in an additive effect, producing 40% lysates from the indicated breast cancer cell lines were subjected to growth inhibition of T47D cells. In contrast, AGN194204 western blot analysis using the anti-PPAR, anti-RAR, anti-RXR, and was a highly effective antiproliferative compound (70% anti-β-actin antibodies shown on the left. Relative expression of each growth inhibition) when used as a single agent on MDA- protein was determined using the same membrane. (b) Human RXR-α protein was immunoprecipitated (IP RXRα) from the indicated breast MB-468 cells, which express higher levels of RXR-α (Fig. cancer cell lines. Immunoprecipitated complexes were subjected to 2b). Similar results were obtained using SKBR3 cells (data western blotting using anti-PPAR-α and anti-PPAR-γ antibodies. Blots not shown). LA treatment of MDA-MB-468 cells resulted in were stripped and incubated with anti-RXR-α antibody to determine the 20% growth inhibition when used as a single agent, but relative amounts of immunoprecipitated protein in each lane. These because of the magnitude of the AGN194204 effect we experiments were performed three times, with similar results. Repre- sentative blots are shown. (c) An RXR-selective compound potentiates did not observe additional antiproliferative activity when transcriptional activation by a PPAR ligand. The indicated human breast both ligands were used together. cancer cell lines were transiently transfected with a heterologous PPAR-responsive promoter/reporter construct and treated with 100 To determine whether LA and AGN194204 inhibited S- µmol/l of the PPAR ligand γ-linolenic acid (LA), 100 nmol/l of the pan RAR agonist all-trans retinoic acid (RA), 100 nmol/l of the RXR-selec- phase progression in human breast cancer cell lines, we tive ligand AGN194204 (AGN), γ-linolenic acid and retinoic acid performed BrdU incorporation analysis. As shown in Fig. (LA+R), or γ-linolenic acid plus AGN194204 (LA+A) for 24 hours 2c, LA treatment reduced the number of BrdU positive cells before determination of promoter activity. Promoter activity was repre- by 30–50%. AGN194204 had little effect on BrdU incor- sented as relative light units from the luciferase reporter. These experiments were performed three times with similar results. Error bars poration in T47D cells, but it inhibited S-phase progression indicate the standard error of the mean. by 70% in the MDA-MB-468 line. These data indicate that R549 Available online http://breast-cancer-research.com/content/6/5/R546

Figure 2

Greateroid X receptor peroxisome (RXR)- proliferator-activatedα-overexpressing human receptor breast (PPAR)-selective cancer cell lines antiproliferative and apoptotic responses potentiated by AGN194204 in retinoid X receptor (RXR)-α-overexpressing human breast cancer cell lines. (a) Triplicate cultures of estrogen receptor positive/retinoic acid (RA)-sensitive T47D cells were treated for 6 days with vehicle (con), 100 µmol/l of the PPAR-γ ligand γ-linolenic acid (LA), 100 nmol/l AGN194204 (AGN), or both compounds simultaneously (LA+AGN). Proliferation was monitored by cell counting at 2-day intervals using a hemacytometer. (b) Triplicate cultures of estrogen receptor negative/RA-resistant MDA-MB-468 cells were treated for 6 days with vehicle (con), 100 µmol/l LA, 100 nmol/l AGN194204, or both compounds simultaneously (LA+AGN). Proliferation was monitored by cell counting at 2-day intervals using a hemacytometer. (c) LA and AGN194204 inhibit S-phase progression in human breast cancer cell lines. The indicated cell lines were treated with 100 µmol/l LA or 100 nmol/l AGN194204 (AGN). The percentage of S-phase cells was determined by bromodeoxyuridine (BrdU) incorporation assay. (d) PPAR- and RXR-selective ligands alter expression of nonoverlapping sets of cell cycle regulatory proteins. Whole cell lysates from the indicated human breast cancer cell lines treated with 100 µmol/l LA for up to 24 hours were subjected to western blot with the antibodies shown on the left. Relative expression of each protein was determined using the same membrane. (e) Whole cell lysates from the indicated human breast cancer cell lines treated with 100 nmol/l AGN194204 for up to 24 hours were subjected to western blot with the antibodies shown on the left. Relative expression of each protein was determined using the same membrane. R550 Breast Cancer Research Vol 6 No 5 Crowe and Chandraratna

the RXR-selective ligand has substantial antiproliferative PPAR-α/RXR synergy results in apoptotic effects on activity in vitro against breast cancer cell lines expressing human breast cancer cell lines higher levels of RXR-α. In contrast to the effects of LA, treatment of breast cancer cell lines with 100 nmol/l of the PPAR-α selective ligand To determine changes in expression of genes that regulate HETE resulted in nuclear condensation, cell rounding, and cell cycle progression resulting from LA treatment of human detachment from the culture dish, which is indicative of breast cancer cell lines, we examined protein levels by apoptosis (data not shown). To confirm this interpretation, western blot. As shown in Fig. 2d, most alterations in the we performed TUNEL assays to measure apoptotic cell expression of these genes did not occur until 16 hours after death in vitro. As shown in Fig. 3a, HETE treatment resulted LA exposure. Cyclin E levels decreased by twofold at this in 35–50% TUNEL-positive cells after 24 hours. Treatment time point in T47D cells and was undetectable in the MDA- with 100 nmol/l AGN194204 alone did not produce signif- MB-468 line. Cdk1 levels also decreased in both cell lines icant increases in the number of apoptotic cells. However, by the 16-hour and 24-hour timepoints. By 24 hours, mark- treatment with a combination of HETE and AGN194204 edly decreased protein levels of a number of cell cycle reg- resulted in 50–75% TUNEL-positive cells after 24 hours. ulatory genes were noted, including ERK1, cyclin A, c-myc, This effect was more pronounced in SKBR3 and MDA-MB- and JunB. The majority of these genes are involved in regu- 468 cells, which express higher levels of RXR-α protein. lating S-phase progression in many cell types. Regulation These results indicate that selective PPAR activation can

of G2 phase proteins such as cyclin B were unaffected by produce different cellular responses in human breast can- LA treatment (data not shown). These results indicate that cer cell lines and that the RXR-selective ligand can poten- LA-induced changes in gene expression correlate with tiate both antiproliferative and apoptotic effects. delayed S-phase progression, as shown by BrdU incorporation analysis in human breast cancer cell lines. To determine whether HETE-mediated apoptosis in human breast cancer lines was the result of intrinsic or extrinsic Treatment with the RXR-selective ligand AGN194204 pathways, we examined patterns of caspase activation in altered expression of a larger group of genes, including these cells. As shown in Fig. 3b, caspase 10 activation in those encoding growth factor receptors, mitogen-activated response to HETE (as shown by decreased immunoreactiv- protein kinases, cyclins, and Cdks. As shown in Fig. 2e, ity to the inactive proenzyme) was observed by 8 hours in expression of epidermal growth factor receptor and the both T47D and MDA-MB-468 cells. Activation of the down- scatter factor receptor c-met, which are overexpressed in stream effector caspase 3 was evident by 8–16 hours in MDA-MB-468 cells, was markedly inhibited by 16 hours of MDA-MB-468 cells. Caspase 9 expression was below the exposure to AGN194204. Decreased expression of these detection limit for western blot in this cell line. Activation of receptors in MDA-MB-468 cells correlated with reduced caspases 3 and 9 was not evident until 24 hours in T47D levels of downstream signaling proteins in the mitogen-acti- cells, which correlated with slightly decreased numbers of vated protein kinase pathway such as ERK1 and ERK3. In apoptotic cells at this time point. Activation of caspases in contrast, levels of these proteins were largely unaffected in the extrinsic (death receptor mediated) pathway such as T47D cells, which are less sensitive to the antiproliferative caspase 8 was not observed following HETE treatment effects of AGN194204. Cyclin B protein levels were also (data not shown). These results indicate that selective acti- decreased by 16 hours in the MDA-MB-468 line, but were vation of PPAR-α activates the intrinsic apoptotic pathway largely unaffected in T47D cells except for a transient in human breast cancer cell lines. decrease observed at 8 hours. Expression of cyclin E, Cdk1, Cdk6, and Fra-1 was inhibited by twofold to fourfold RXR-α overexpression in human breast cancer cell lines in both cell lines by 8 hours; these genes represent the ear- results in phenotypic sensitization to the RXR-selective liest observed changes in response to AGN194204 in ligand these cell lines. In contrast, Cdk4 levels were largely unaf- Our results suggest that RXR-α overexpression may sensi- fected by AGN194204 in either cell line, indicating that the tize cells to the antiproliferative effects of AGN194204 and observed changes in gene expression were specific. There- potentiate additional PPAR-regulated responses in human fore, although some of the AGN194204-induced changes breast cancer cell lines. To test this hypothesis we overex- in gene expression overlapped with those regulated by LA, pressed RXR-α by stable transfection in MCF7 and T47D others were specific for each selective ligand. These data cells, which normally express low levels of the receptor. indicate that increased sensitivity of RXR-α overexpressing RXR-α protein levels in these stable clones are shown in human breast cancer cell lines to AGN194204 is reflected Fig. 4a. To determine whether these clones were sensitized in changes in gene expression. to the antiproliferative effects of AGN194204, we performed proliferation assays using ligand or vehicle treated cells. As shown in Fig. 4b, treatment with 100 nmol/l AGN194204 inhibited proliferation of RXR-α R551 Available online http://breast-cancer-research.com/content/6/5/R546

Figure 3 Figure 4

Theandbreast hydroxyeicosatetraenoicperoxisome cancer cell proliferator-a lines, which acidctivated is potentiated (HETE) receptor induces by (PPAR)- AGN194204 apoptosisα-selective in human lig- and hydroxyeicosatetraenoic acid (HETE) induces apoptosis in human breast cancer cell lines, which is potentiated by AGN194204. (a) The indicated breast cancer cell lines were treated with vehicle (con), 100 µmol/l HETE, 100 nmol/l AGN194204 (AGN), or both compounds simultaneously (HETE+AGN) for 24 hours. The percentage of apoptotic cells was determined by dUTP nick-end labeling (TUNEL) assay and fluorescence microscopy. These experiments were performed three times, with similar results. Error bars indicate the standard error of the mean. (b) The PPAR-α-selective ligand HETE activates the intrinsic apoptotic pathway in human breast cancer cell lines. Whole cell lysates from the indicated breast cancer cell lines treated with 100 µmol/l HETE for up to 24 hours were subjected to western blot, with anti-caspase (casp) and anti-β-actin antibodies shown on the left. Relative Retinoidantiproliferativelines X receptor and (RXR)-apoptoticα overexpression responses in human induces breast ligand-selective cancer cell expression of each protein was determined using the same membrane. antiproliferative and apoptotic responses in human breast cancer cell (a) These experiments were performed three times, with similar results. lines. RXR-α expression in T47D and MCF7 G418 resistant control Representative blots are shown. clones (neo) and RXR-α stable clones (RXR-1, RXR-2). Whole cell lysates were subjected to western blot with anti-RXR-α and anti-β-actin antibodies. These experiments were repeated three times with inde- overexpressing T47D clones by 50%. Slightly greater inhi- pendent clones. Representative blots are shown. (b) RXR-α overexpression induces antiproliferative response to the RXR-selective ligand bition of proliferation (55%) by AGN194204 was observed AGN194204 in T47D stable clones. Triplicate cultures of G418 resist- in RXR-α overexpressing MCF7 clones, reflecting higher ant control cells (neo) and RXR-α overexpressing clones (RXR1) were receptor expression in these cells (Fig. 4c). In contrast, treated with vehicle or 100 nmol/l AGN194204 (AGN) for 6 days. Cells AGN194204 exposure had less effect on G418 resistant were counted at 2-day intervals using a hemacytometer. (c) RXR-α control clones. AGN194204 also increased the percent- overexpression induces antiproliferative response to the RXR-selective ligand AGN194204 in MCF7 stable clones. Triplicate cultures of G418 age of apoptotic cells in cultures of RXR-α overexpressing resistant control cells (neo) and RXR-α overexpressing clones (RXR1) clones exposed to HETE. As shown in Fig. 4d, were treated with vehicle or 100 nmol/l AGN194204 (AGN) for 6 days. AGN194204 increased the percentage of TUNEL-positive Cells were counted at 2-day intervals using a hemacytometer. (d) RXR- cells by more than 50% in HETE-treated RXR-α overex- α overexpression potentiates the hydroxyeicosatetraenoic acid (HETE) apoptotic response to the RXR-selective ligand AGN194204 in T47D pressing clones. In contrast, treatment with RXR-selective and MCF7 clones. G418 resistant control cells (neo) and RXR-α over- ligand increased the percentage of TUNEL positive cells by expressing clones (RXR-1, RXR-2) were treated with vehicle (con), 100 only 20% in HETE treated control clones. Treatment with µmol/l HETE, 100 nmol/l AGN194204, or both compounds simultane- AGN194204 as a single agent had minimal effect on the ously (H+AGN) for 24 hours. The percentage of apoptotic cells was determined by dUTP nick-end labeling (TUNEL) assay using fluores- number of apoptotic cells in cultures of these clones. These cence microscopy. These experiments were performed three times, results indicate that RXR-α overexpression is sufficient to with similar results. Error bars indicate the standard error of the mean. R552 Breast Cancer Research Vol 6 No 5 Crowe and Chandraratna

sensitize breast cancer cell lines to the antiproliferative particular interest is the increased sensitivity to this ligand effects of AGN194204 and potentiates the PPAR-α medi- of cell lines expressing higher levels of RXR-α. Some of ated apoptotic response. these estrogen receptor negative cell lines have been shown to be resistant to the antiproliferative effects of the Discussion RAR agonist all-trans RA [39]. The RXR ligand mediated One of the key findings of this study is that PPARs can effects were associated with inhibition of growth factor sig- mediate selective responses in human breast cancer cell naling and cell cycle regulatory pathways. RXR-α overex- lines, namely growth inhibition and apoptosis. Our data pression was sufficient to reproduce the effects of the indicate that PPAR-α, when activated by the selective lig- selective ligand in MCF7 and T47D cells. The effects of and HETE, induces apoptosis in breast cancer cell lines. AGN194204 on breast cancer cell lines suggest that this This was characterized by activation of caspases 9 and 10 ligand may have chemopreventive or chemotherapeutic followed by caspase 3. Activation of caspase 8 was not effects in vivo. In previous studies, synthetic RXR ligands observed in cells treated with HETE. Future experiments have been effective against mammary carcinogenesis in will characterize downstream targets of activated caspases animal models [33]. However, a phase II clinical trial of oral in HETE-treated cells. Conversely, LA-mediated activation bexarotene showed limited antitumor effects in patients of PPAR-γ resulted in decreased growth characterized by with metastatic breast cancer [36]. S-phase inhibition. PPAR-γ activation resulted in changes in expression of a specific set of cell cycle regulatory Our findings also demonstrate that AGN194204 potenti- genes. Previous studies have indicated that PPAR-γ mediated the antitumor effects of PPAR ligands. Combination of ates the action of LA in breast cancer cell lines [15]. These PPAR and RXR ligands increased transactivation of a cells have also been shown to contain a functional PPAR responsive promoter in human breast cancer cell lines, response [14]. In addition to HETE and LA, studies have which is in agreement with previous studies [14]. These shown that a variety of fatty acids can act as ligands for results suggest that multidrug regimens consisting of PPARs, with disparate effects on promoter transactivation PPAR and RXR ligands may provide a therapeutic advan- [16]. This large group of natural ligands can complicate the tage in breast cancer treatment. The molecular mecha- interpretation of studies examining the risk for breast can- nisms regulating these effects are currently unclear. PPAR cer development associated with high-fat diets. The results and RXR ligands have been shown to differentially recruit of our experiments suggest that PPAR ligands may affect subsets of transcriptional coactivator proteins to the recep- established breast cancer cell lines differently than normal tor complex [40]. Combinations of these ligands may result mammary epithelium in animal or human studies. In the rat in additional coactivator recruitment, leading to enhanced mammary carcinogenesis model, a synthetic PPAR-γ ligand transcriptional activation and cellular effects. The molecular inhibited cancer development [18]. Clarification of these characterization of these pathways in human breast cancer effects awaits further experimentation. cells and testing the effects of PPAR and RXR ligands on mammary carcinogenesis in coactivator-null mutant mice Activation of PPAR-γ has been shown to have multiple anti- will be important in future studies. tumor effects on breast cancer cells (for review, see Ker- sten and coworkers [38]). PPAR-γ activation using Conclusion synthetic ligands such as TZDs was shown to induce lipid PPAR and RXR proteins are expressed by human breast accumulation in and differentiation of breast cancer cell cancer cell lines. These proteins heterodimerize and trans- lines [17]. In the present study we did not observe these activate a PPAR-responsive promoter. The magnitude of types of changes; rather, PPAR-γ activation resulted in anti- transactivation was greater in breast cancer cell lines proliferative effects. These differences may be due to the expressing higher levels of RXR-α. PPAR-α and PPAR-γ fact that different breast cancer cell lines were used in the ligands induced apoptotic and antiproliferative responses two studies. The role of PPAR-α ligands as a chemopre- in these lines, respectively, which were associated with ventive or chemotherapeutic agent for breast cancer is less specific changes in gene expression. These responses clear. However, our studies indicate that PPAR-α activation were potentiated by the RXR-selective ligand by HETE is a potent inducer of apoptosis in established AGN194204. RXR-α overexpressing RA-resistant breast human breast cancer cell lines. In vitro studies are inher- cancer cell lines were more sensitive to the effects of the ently limited in determining the effects of PPAR activation RXR-selective compound. Forced RXR-α overexpression on clinical breast cancer, given that a phase II trial of trogl- in other breast cancer cell lines resulted in greater sensitiv- itazone showed little effect on refractory tumors [19]. ity to the selective ligand.

Another important finding of the present study is the anti- Competing interests proliferative effect of the synthetic RXR ligand RASC is a research director at Allergan, which developed AGN194204 on human breast cancer cell lines. Of the RXR-selective ligand AGN194204. DLC has acted as R553 Available online http://breast-cancer-research.com/content/6/5/R546

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